Interactions between Casein Kinase Ie (CKIe) and Two
Substrates from Disparate Signaling Pathways Reveal
Mechanisms for Substrate-Kinase Specificity
Caroline Lund Dahlberg1, Elizabeth Z. Nguyen2, David Goodlett2, David Kimelman1*
1 Department of Biochemistry, University of Washington, Seattle, Washington, United States of America, 2 Department of Medicinal Chemistry, University of Washington,
Seattle, Washington, United States of America
Abstract
Background: Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways.
The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini.
Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms
often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we
have examined the interaction between CKIe and two substrates from different signaling pathways.
Methodology/Principal Findings: CKIe, but not CKIa, binds to and phosphorylates two proteins: Period, a transcriptional
regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GSTpull-down assays data to show that two key residues in CKIa’s kinase domain prevent Disheveled and Period from binding.
We also show that the unique C-terminus of CKIe does not determine Dishevelled’s and Period’s preference for CKIe nor is it
essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIe with its substrates. We
demonstrate that autophosphorylation of CKIe’s C-terminal tail prevents substrate binding, and use mass spectrometry and
chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase
domain prevents substrate phosphorylation and binding.
Conclusions/Significance: The biochemical interactions between CKIe and Disheveled, Period, and its own C-terminus lead
to models that explain CKIe’s specificity and regulation.
Citation: Dahlberg CL, Nguyen EZ, Goodlett D, Kimelman D (2009) Interactions between Casein Kinase Ie (CKIe) and Two Substrates from Disparate Signaling
Pathways Reveal Mechanisms for Substrate-Kinase Specificity. PLoS ONE 4(3): e4766. doi:10.1371/journal.pone.0004766
Editor: Wenqing Xu, University of Washington, United States of America
Received January 6, 2009; Accepted February 10, 2009; Published March 10, 2009
Copyright: ß 2009 Dahlberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH HD27262 (to DK) T32-GM07270 (to CLD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
phosphorylation is accepted as an indicator of Dsh’s activation in
both pathways [18–30]. During canonical Wnt signaling, phosphorylated Dsh induces the translocation of Wnt responsive
signalosomes to the cell membrane, as an early step in stabilizing
the transcriptional coactivator, b-catenin [26–31]. Dsh’s phosphorylation also indicates its activation in Frizzled/PCP signaling
[2,19,21–33]. Although CKIa, CKIc, and CKIe have all been
implicated in Wnt and/or Frizzled/PCP signaling, only CKIe has
been shown to bind to and phosphorylate Dsh, in vivo. Despite the
similarity between the kinase domains of CKIe and the other CKI
isoforms, CKIe is the kinase required for the full activation of Dsh
and its downstream effects [19,23,27].
Physiological outputs (hormonal, metabolic and cellular) that
are entrained to the 24 hour cycle of the earth’s rotation have
been described in organisms from cyanobacteria through humans,
and are based on molecular clockwork, [reviewed in 3,34,35]. The
cyclic expression and localization of the transcriptional regulator,
Period (Per), is in large part regulated by phosphorylation by CKIe
in mammals [36–40]. There are three Per isoforms in mice
(mPer1, mPer2, mPer3) that display different expression and
regulation patterns [41,42]. Mutations in CKIe disrupt the
Introduction
CKI family members have been implicated in a wide range of
signaling activities including involvement in the Wnt, planar cell
polarity (PCP) and circadian rhythms pathways [1–3]. In
mammals, the CKI family of serine/threonine kinases consists of
seven distinct genes (a, b, c1, c2, c3, d, e) that code for enzymes
with highly conserved kinase domains and divergent amino- (N-)
and carboxy- (C-) termini. CKIe in particular has a long (,100
residue) C-terminus that undergoes inhibitory autophosphorylation at conserved serine and threonine residues [4–7]. Subcellular
localization of different isoforms in some cases is determined by
splice variants (in the case of CKIa) [8,9] or fatty-acylation (in the
case of CKIc) [10], but Western and Northern blotting
experiments, and in situ hybridization studies indicate that the
a, d, and e isoforms have generally overlapping expression [9,11–
14]. It is therefore interesting that despite high sequence similarity
and common expression patterns, CKI family members have
different targets in the cell [10,15–18].
Dishevelled (Dsh) is a 100 kDa protein that is required for
canonical Wnt and Frizzled/PCP signaling [19,20–23]. HyperPLoS ONE | www.plosone.org
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CKIe Interactions
circadian pathway through aberrant or reduced phosphorylation
of Per [39,43–45]. Both CKIe and CKIa isoforms are found in
cells that exhibit circadian cycling, including the master circadian
oscillator in mice, the suprachiasmatic nucleus [14]. Why one CKI
isoform is active in the pathway while the other is not has not been
investigated.
Per and Dsh bind specifically to CKIe [19,37,46–48]. To
determine how CKIe and CKIa differ with respect to binding by
substrates, we performed a series of in vitro experiments to probe
the direct interaction between CKIe and two substrates from
disparate signaling pathways. We show that, surprisingly, features
C-terminal to the kinase domain of CKIe are not responsible for
the difference in binding of the substrate proteins to the two CKI
kinases. Instead, two residues on the kinase domain of CKIe
determine Dsh’s and Per’s affinity for CKIe, whereas the Cterminus of CKIe stabilizes interactions between CKIe and Dsh
and Per. Finally, we show that autophosphorylation of CKIe’s Cterminus inhibits substrate binding. Using chemical cross-linking
and tandem mass spectrometry, we demonstrate that autophosphoryation of the C-terminal tail changes its interaction with the
kinase domain, revealing why the autophosphorylated tail inhibits
substrate phosphorylation and binding.
developed an expression system for large quantities of pure fulllength Xenopus CKIe fused to GST (CKIe-GST; see Methods).
Using a similar strategy we also expressed full-length Xenopus CKIa
fused to GST (CKIa-GST). Both kinases were catalytically active
against b-catenin, indicating that they were both well folded (data
not shown). Because CKIe autophosphorylation regulates substrate binding (see below), we used dephosphorylated CKIe for our
binding studies except where otherwise noted. Using GST-pulldown assays we found that 35S-labelled Xenopus Dsh (xDsh) and
mouse Per1 (mPer1) bound specifically to CKIe compared to
CKIa (Fig. 1), recapitulating previously published in vivo results
[19,37,46–48]. This provided us with a useful assay system to
examine CKIe specificity for these substrates.
CKIe does not require its C-terminus to interact with
substrate proteins
CKIe and CKIa are 75% identical, and 89% similar in their
kinase domains (Fig. 2). This high degree of conservation between
the kinases suggested that full binding by mPer1 and xDsh to
CKIe might depend on the long C-terminal tail, which is unique
to CKIe. We first examined binding of mPer1 and xDsh to
CKIeDC, which truncates the kinase at residue 319 (Fig. 2).
Figure 3A shows that xDsh and mPer bound to CKIe and
CKIeDC to the same extent, demonstrating that CKIe does not
require the autophosphorylation region of the C-terminus to bind
to substrate proteins.
Because truncation of CKIe at residue 319 did not diminish
xDsh or mPer1’s affinity for the kinase, another part of the protein
must determine CKIe’s interaction with substrate proteins.
CKIeDC retains 22 residues that are not conserved between
CKIe and CKIa (Fig. 2) and these could contribute to the
substrate-kinase interaction. We produced CKIeDDC, which is
truncated at residue 295 and therefore even more closely
resembles CKIa (Fig. 2). Figure 3B shows that CKIeDC and
Results
CKIe, but not CKIa, binds to xDsh and mPer1 in vitro
Per1 and Dsh bind to CKIe in vivo, but they do not interact
with the related isoform CKIa [23,47–49]. In order to determine
if these substrates bind to the two kinases with the corresponding
affinities in vitro, we used a Glutathione S-Transferase (GST) pulldown system to follow protein-protein interactions. Due to protein
instability and toxicity in E. coli, prior in vitro studies of CKIe have
used a C-terminally truncated form of the protein called CKIeDC
[48,50,51], which eliminates the last 98 residues of the protein.
Since we wanted to include all of the protein in our analysis, we
Figure 1. Recombinant CKIe, but not CKIa, interacts with mPer1 and xDsh. (A) GST, GST-CKIe, or GST-CKIa was bound to glutathione
sepharose, and then incubated with 35S-labeled mPer1 or xDsh. 10% of the mPer1 input and 25% of the xDsh input are shown. Coomassie stained gel
shows the levels of GST fusion protein used for each pull-down. (B) Quantification of three independent experiments. Values are normalized against
the amount of protein bound by GST-CKIe, and error bars represent standard deviation of the mean.
doi:10.1371/journal.pone.0004766.g001
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conserved between CKIe and CKId but are not conserved in
CKIa. Two residues that are conserved in CKIe and CKId but
not CKIa, and have solvent exposed side-chains, Asparagine 275
(N275) and Arginine 279 (R279), are shown in red (Fig. 4A). In
CKIa, the corresponding residues have chemical properties that
differ substantially from the CKIe residues (AspRIle and
ArgRThr), and therefore these residues could contribute specificity to CKIe-substrate interactions.
We initially changed N275 and R279 to alanines in the context
of the conventional CKIeDC truncation (CKIeDC N275A/
R279A, Fig. 2). These mutations significantly disrupted the
binding of xDsh to CKIe, although they had no effect on mPer1
binding (Fig. 4B, lane 4, and Fig. 4C). However, when we replaced
N275 and R279 with the corresponding CKIa residues Ile and
Thr, respectively (CKIeDC N275I/R279T, Fig. 2), the binding
between CKIe and mPer1 was abolished and the binding between
CKIeDC and xDsh was further reduced (Fig. 4B, lane 5, and
Fig. 4C). In conclusion, N275 and R279 are required for strong
binding between CKIe and xDsh, whereas the corresponding
chemically distinct residues in CKIa prevent binding of mPer1 to
CKIe.
Figure 2. Protein constructs used in this work. CKIa and -e are
shown with their conserved kinase domains in gray and black,
respectively (89% similarity, 75% identity). The arrow indicates the
position of residue 295 in CKIe, and the non-conserved, charged region
of the protein is colored red. The filled arrowhead indicates the position
of residue 319, where CKIe is conventionally truncated. The C-terminus
contains autophosphorylation sites and is colored yellow. The open
arrowhead and white bars indicate the position of residues N275 and
R279 (CKIe), and I283 and T287 (CKIa).
doi:10.1371/journal.pone.0004766.g002
The C-terminus of CKIe modulates the effect of
mutations in the kinase domain
CKIeDDC each bound to xDsh or mPer1with the same affinity.
CKIe must therefore bind to substrate proteins through its kinase
domain.
In order to determine what role the C-terminus might play in
substrate recognition, we introduced the N275I and R279T
substitutions into full-length CKIe (CKIe N275I/R279T, Fig. 2).
xDsh’s binding to CKIe N275I/R279T is marginally enhanced
compared to its binding to CKIeDC N275I/R279T, while mPer1
binding is completely restored (compare Fig. 4 to Fig. 5). We
conclude that the C-terminus is not necessary for CKIe to bind to
substrates (see Fig. 3), but that it stabilizes otherwise weakened
interactions between xDsh or mPer1 and CKIe.
We constructed a chimeric protein that contains the kinase
domain of CKIa and the C-terminus of CKIe to determine if the
C-terminus of CKIe could confer CKIe-like binding to CKIa
Residues N275 and R295 are responsible for enhanced
substrate binding to CKIe
To identify residues within the CKIe kinase domain that could
confer specificity, we examined the structure of CKId. CKId is
considered a very close relative of CKIe since they share 82%
identity over the entire protein and 96% identity in their kinase
domains, and since their cellular activities are indistinguishable
[11,13,24,48,52]. Residues shown in cyan in Figure 4A are
Figure 3. xDsh and mPer1 do not require CKIe’s C-terminus for binding. (A, B) Purified GST, GST-CKIe, GST-CKIeDC or GST-CKIeDDC were
bound to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1 as indicated.
doi:10.1371/journal.pone.0004766.g003
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Figure 4. Residues 275 and 279 regulate binding to xDsh and mPer1. (A) Space-filling representation of CKId (PDB ID 1CKJ, [50]). Residues
shown in cyan and red are conserved between CKIe and CKId, but not CKIa. Red residues N275 and R279 are solvent accessible and are chemically
distinct in CKIa. Orange shading shows the position of the ATP binding cleft. (B) Binding of 35S-labeled mPer1 and xDsh to GST-CKIeDC, GST-CKIeDC
N275A/R279A, or GST-CKIeDC N275I/R279T was performed. (C) Quantification of three independent experiments. Values are normalized against the
amount of protein bound by GST-CKIeDC.
doi:10.1371/journal.pone.0004766.g004
and reveals that mPer1 requires an additional CKIe-like environment elsewhere on the kinase in order to bind CKIa.
(CKIaRe, Fig. 2). Neither xDsh nor mPer1 showed strong
binding to the chimeric protein, compared to CKIe (Fig. 6A).
Their weak binding to CKIaRe along with the data presented
above indicates that the C-terminus is able to contact and partially
stabilize the interaction between CKIe and its substrates, but it is
not sufficient for the binding of mPer1 or xDsh to CKI.
Phosphorylation of the C-terminus of CKIe inhibits the
binding of target proteins
Our data shows that the C-terminus of CKIe stabilizes some
substrate interactions, specifically in the absence of motifs that are
required for binding (Fig. 6A and B). However, autophosphorylation of the C-terminus has also been shown to inhibit CKIe’s
activity towards protein targets [6,7,53,54]. We hypothesized that
this effect might be partially mediated by a change in affinity for
substrates. When CKIe is allowed to fully autophosphorylate,
neither mPer1 nor xDsh bind to it (Fig. 7). In addition,
hyperphosphorylated CKIe no longer binds to mAxin, a protein
that is able to bind to both CKIa and CKIe. Hyperphosphorylation of CKIe’s C-terminus thus inhibits the binding of
substrate proteins, and may act as a regulatory mechanism to
control phosphorylation of targets.
Conversion of CKIa to an xDsh-binding protein
We hypothesized that CKIa’s residues I283 and T287, which
correspond to CKIe’s N275 and R279, were responsible for
preventing interactions with CKIaRe. We therefore engineered
CKIaRe I283N/T287R, which is nearly identical to CKIaRe but
contains two point mutations that could potentially enhance binding
by xDsh and mPer1 (Fig. 2). Impressively, conversion of CKIa
residues I283 and T287 to the CKIe identity enabled xDsh to fully
interact with the kinase. In contrast, mPer1 did not bind CKIaRe
I283N/T287R. This data clearly demonstrates that xDsh depends
on the CKIe residues N275 and R279 for strong binding to CKIe,
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Figure 5. The C-terminal tail enhances binding of xDsh and mPer1 to mutant CKIe. (A) GST, GST-CKIe, or GST-CKIe N275I/R279T was bound
to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1. (B) Quantification of three independent experiments. Values are normalized
against the amount of protein bound by GST-CKIe.
doi:10.1371/journal.pone.0004766.g005
Shrimp Alkaline Phosphatase (SAP) after crosslinking but before
trypsin cleavage. SAP treatment reduced the level of phosphorylation of crosslinked CKIe as seen by radioactive labeling on the
protein (data not shown).
Mass spectrometry analysis showed that the sequence PEDLDRERREHDREER is crosslinked to the kinase domain (Fig. S1
and Fig. S2). PEDLDRERREHDREER corresponds to the 20
amino-acid stretch that was removed from CKIeDC to make
CKIeDDC (Fig. 2). Two crosslinks along the PEDLDRERREHDREER peptide position it directly along the basic groove that abuts
the active site of CKIe, which was postulated to be a possible
phosphate recognition region [50,55–60] (Fig. 8B). Lysines 130
and 232 are part of the kinase domain and were crosslinked to
opposite ends of the peptide (PEDLD and EHDREER, respectively). We did not identify any crosslinks in the rest of the Cterminus. This is in agreement with our PAGE results, which
indicate that the crosslinked protein sample is highly heterogeneous and would have reduced abundance of unique cross-linked
peptides (Fig. 8A).
We also collected spectra for two phosphorylated peptides that
were not crosslinked efficiently. These peptides, MGQLRGS
(p)AT(p)RALPPGPPAGAAPNR and ISASQAS(p)VPFDHLGK
(Fig. S3 and Fig. S4) are phosphorylated at previously described
autoinhibitory autophosphorylation sites [6]. The two phosphorylated peptides were found even in the sample treated with SAP.
Presumably dephosphorylation of these tryptic peptides was
blocked due to inefficient contacts between SAP and the
phosphates, suggesting that the phosphate modifications may be
protected through interactions with the rest of CKIe.
Autophosphorylation of the C-terminus of CKIe stabilizes
its interaction with the kinase domain
Despite numerous attempts, we were unable to crystallize fulllength autophosphorylated CKIe, raising the possibility that the
phosphorylated C-terminus does not adopt a single conformation,
which is necessary to obtain a crystal. It was possible, however,
that the autophosphorylated C-terminus of CKIe adopts a stable
set of conformations that blocks the binding site for substrates. As
an alternative approach to crystallography, we performed a set of
crosslinking and mass spectrometry experiments to establish how
autophosphorylation of CKIe’s C-terminus interferes with substrate binding.
We first looked for evidence of a conformational change by
following differences in electrophoretic mobility of purified,
hyperphosphorylated CKIe upon the addition of 1-Ethyl-3-[3dimethylaminopropyl]carbodiimide Hydrochloride (EDC), a zerolength crosslinker. When two or more proteins are crosslinked by
EDC, the expected product is a band of approximately their
additive molecular weights. In the case of CKIe, no high
molecular weight bands appear, demonstrating that CKIe does
not form dimers or higher-order oligomers in solution. Instead, the
single band corresponding to CKIe broadened considerably, and
showed faster mobility after EDC treatment as seen by PAGE
analysis (Fig. 8A, lane 4). Interestingly, unphosphorylated CKIe
did not show a change in mobility after crosslinking and it
continued to migrate as a single band (Fig. 8A, lane 2). The broad
band that appears after crosslinking suggests that the autophosphorylated CKIe occupies multiple conformations.
We investigated where CKIe’s hyperphosphorylated C-terminus binds to the kinase domain using mass spectrometry on three
different trypsin-digested preparations of phosphorylated and then
crosslinked protein. In order to alleviate potential problems with
phosphorylation inhibiting tryptic cleavage or detection of
peptides on the mass spectrometer, we treated one sample with
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Discussion
CKIe and CKIa are closely related kinases with very divergent
C-termini. We show here that recombinant CKIe and CKIa
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Figure 6. A. Neither xDsh nor mPer1 bind strongly to CKIaRe. GST, GST-CKIe (partially purified) or GST- CKIaRe, was bound to glutathione
sepharose and incubated with 35S-labeled xDsh or mPer1. (B) GST, GST-CKIe, or GST- CKIaRe I283N/T287R, was bound to glutathione sepharose and
incubated with 35S-labeled xDsh or mPer1. The bar graphs represent quantification of three independent experiments. Values are normalized against
the amount of protein bound to GST-CKIe.
doi:10.1371/journal.pone.0004766.g006
interact in vitro with two known CKIe substrates similarly to the
previously published in vivo interactions between endogenous
proteins [23,47–49]. Using recombinant proteins, we demonstrate
that CKIe’s C-terminus is not essential for binding to xDsh and
mPer1, and show that two residues in the conserved kinase
domain, N275 and R279, provide a critical interface for binding
by these substrates. Additionally, we show that the C-terminus of
CKIe can positively regulate substrates by stabilizing contacts with
the kinase domain when dephosphorylated, whereas it can
negatively regulate substrate-kinase interactions when autophosphorylated. Finally, we show that a conformational change occurs
in the C-terminus of CKIe such that the C-terminus probably
binds near the active site when the kinase is autophosphorylated.
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Selectivity of CKIe binding for Dishevelled
The binding of xDsh to CKIe and not CKIa primarily requires
the residues N275 and R279 (Fig. 9A). Mutation of these residues
to either alanines or the CKIa identities, isoleucine and threonine
respectively (Fig. 9B), prevented binding. Thus, the identity of
these residues is essential, suggesting that Dsh directly contacts
these residues. We also noted that the extent to which Dsh was
phosphorylated (visualized by a change in electrophoretic
mobility, Fig. 4) was different depending on the mutations that
were made. The identities of the residues at positions 275 and 279
in CKIe may therefore also be important for correctly positioning
Dsh for full phosphorylation. The C-terminal tail of CKIe
provided some enhancement of xDsh binding and increased the
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Since the F-X-X-X-F motif binds both CKIa and CKIe, it
contributes to the binding affinity of mPer1 to CK1 but it is not
involved in the selectivity. Thus, we conclude that another residue
(or residues) on CKIe besides N275 and R279 contributes to the
selectivity (Fig. 9A). This additional residue (or residues) could be
amino acids that in CKIe positively enhance the binding of mPer1,
or residue (or residues) that in CKIa repel mPer1. Nonetheless,
our results demonstrate that the lack of the tail and changes in two
residues in CKIa are sufficient to explain the lack of binding of
CKIa to mPer1.
The role and structure of the C-terminal tail
The C-terminus of CKIe has been shown in previous reports to
be catalytically autoinhibitory and important for regulation within
signaling pathways [61–63]. Wnt signaling lowers the level of
autophosphorylation on CKIe’s C-terminus [63] though only a
subset of the sites are phosphorylated in vivo, judging by
polyacrylamide gel and isoelectric focusing data. Our data show
that phosphorylation of the C-terminus blocks interactions
between CKIe and binding partners, and it is therefore likely
that some portion of the lowered phosphorylation activity seen in
vivo is due to less efficient substrate binding.
The C-terminus of CKIe has been proposed to act as a pseudosubstrate for the kinase during autoinhibition [4,50,57]. The
positively charged groove in front of the active site has been
postulated to bind phosphopeptides that helps determine the S(p)X-X-S specificity of the kinase family [1,4,50,55–59,64]. Thus the
phosphates in the tail could occupy that site and prevent substrates
from binding. Our data suggest the alternative view that the
phosphorylation of the tail causes the unphosphorylated but highly
charged peptide, PEDLDRERREHDREER to sit across the front
of the kinase’s active site when the C-terminal tail is autophosphorylated, thus preventing substrate access to the active site
and/or phosphate recognition groove (Fig. 8B and 9E). Intriguingly, two previous structures of CKIdDC failed to show any
density for this peptide [50,58], indicating that phosphorylation of
the C-terminal tail is necessary to lock this peptide into place.
In the model illustrated in Figure 9D, after PEDLDRERREHDREER is directed through the phosphate recognition groove, the
rest of the C-terminus is led to the back of the kinase (Fig. 9D). The
back face is very basic as shown in an electrostatic view (Fig. 8B),
providing many sites for the phosphates in the tail to interact. The
heterogeneity of the cross-linked phosphorylated CKIe and the lack
of identification of prominent cross-linked tail peptides suggests that
the interaction of the tail with the back side is highly variable, which
also explains our inability to crystallize full-length CKIe (the same
was also found with full-length CKId [50,58]). Once at the back side
of the kinase domain, some phosphorylated residues on the Cterminus are buried in the basic face to stabilize the conformation
(Fig. 9E). The long C-terminus could then fold against the back side
of the kinase domain to occlude the binding sites for Dsh, Per and
Axin. Thus, we suggest that phosphorylation of the tail has two
roles, to bring the proximal C-terminus in front of the active site to
prevent substrate phosphorylation and to prevent substrate binding
using the distal part of the tail.
Casein Kinase family members have many targets in the cell.
The results of this study designate two amino acid residues that
determine the ability of two different CKI isoforms to select and
bind their target proteins. Furthermore, our work on auto- and
dephosphorylated CKIe introduces a means to regulate CKIe’s in
the presence of its targets. These two biochemical mechanisms
could help maintain the strict spatial and temporal control of
CKIe and CKIa that is necessary in both the circadian rhythms
and Wnt pathways.
Figure 7. Autophosphorylation of CKIe inhibits binding by
substrate and scaffolding proteins. Purified GST and GST-fusion
proteins were bound to glutathione sepharose and bound GST-CKIe
was incubated with SAP or ATP for 1 hour. Resin was incubated with
35
S-Methionine-labeled xDsh, mAxin or mPer1. 10% of the mPer1and
mAxin input and 25% of the xDsh input were run. Coomassie stained
gel shows the levels of GST fusion protein used for each pull-down.
doi:10.1371/journal.pone.0004766.g007
level of phosphorylation by the mutant protein (Fig. 5), but it was
not sufficient for binding. Strikingly, we saw full binding of xDsh to
CKIa simply by changing the I283 and T287 to the CKIe identity
and adding on the C-terminal tail (Fig. 9C). In the absence of the
C-terminal tail, Dsh did not bind to the mutated CKIa (data not
shown), which strengthens our hypothesis that the C-terminus of
CKIe does contact Dsh, but is not required in for interactions with
wild-type CKIe. These results allow us to fully explain the
selectivity of CKIe for Dishevelled.
Selectivity of CKIe binding for Period
The binding of mPer1 to CKIe is more complex than for
Dishevelled. Changing residues N275 and R279 to alanine in the
context of CKIeDC did not disrupt mPer1 binding, whereas
mutation of these residues to the CKIa identity in CKIeDC
eliminated all binding. This result indicates that the CKIa residues
at this position prevent binding by mPer1, but that the residues
may not be directly involved in its binding (Fig. 9B). This is
supported by the observation that changing these residues in
CKIa to the CKIe identity (as well as adding the C-terminal tail)
did not rescue mPer1 binding even though it completely rescued
xDsh binding.
The C-terminal tail does have an important contribution to
mPer1 binding since full-length CKIe N275I/R279T could fully
bind mPer1 whereas CKIeDC N275I/R279T could not bind
mPer1. Thus the C-terminal tail can override the repulsive effect
of N275I and R279T. However, since the tail did not rescue
CKIaRe I283N/T287R, it alone is not sufficient for binding
mPer1 to CKIa. These results reveal that another site (or sites) on
CKI must also contribute to the difference in binding between
CKIe and CKIa.
One CKI-binding motif, F-X-X-X-F (where F is phenylalanine
and X can be any amino acid residue), is present in mPer1 but not
in xDsh [49]. Studies of the NFAT family of proteins suggest that
the F-X-X-X-F motif enables stable interactions with both CKIa
and CKIe [49]. Mutation of the phenylalanine residues to alanines
abolishes binding by NFAT to CKIa and CKIe. mPer1 shows the
same loss of binding when its F-X-X-X-F motif is mutated [37,49].
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Figure 8. Analysis of the binding of the C-terminal tail. (A) Full-length CKIe was incubated with either SAP or ATP prior to reaction with the
EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIe. In lane 4, there is marked change
in the migration of autophosphorylated, crosslinked CKIe (bracketed). Asterisk shows a high molecular weight species that may correspond to SAPCKIe oligomers (lane 2). (B) Space-filling models of CKId are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish
electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively
charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is
conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows
the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of
the tail onto the backside of the kinase.
doi:10.1371/journal.pone.0004766.g008
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Figure 9. Model of inter- and intra-molecular interactions involving CKIe. Red blocked arrows represent inhibition of binding and green
arrows represent positive binding interactions. (A–D) Back face of the kinase. (A) CKIe binds to Dsh and Per using different binding sites. (B) Mutation
of CKIe N275 and R279 to the corresponding CKIa identity inhibits Dsh and Per binding; however, the C-terminal tail and at least one other residue in
the kinase domain promote Per binding. (C) Changing two residues in CKIa to the CKIe identity along with adding CKIe’s C-terminus enables Dsh to
bind to CKIa. Per is unable to bind this chimeric kinase. (D) Binding of the autophosphorylated tail to the backside prevents the binding of substrates.
Phosphorylated sites detected by mass spectrometry are shown on the tail. (E) View of the front side of the kinase. Left, CKIe’s C-terminus is labile
when it is not phosphorylated, and CKIe is able to bind to partners. Right, upon incubation with ATP, CKIe autophosphorylates and the C-terminus
binds tightly to the back side of the kinase domain. This positions the peptide PEDLDRERREHDREER next to the active site and the phosphate
recognition groove. The X’s show identified crosslinks between the peptide and the kinase domain.
doi:10.1371/journal.pone.0004766.g009
and were induced with 0.5 mM IPTG. Cells were pelleted and
opened by sonication, in the presence of PMSF, Leupeptin and
Pepstatin A. Lysate was cleared by centrifugation, and was applied
to Glutathione Sepharose 4B resin (Amersham) or Glutathione
Sepharose resin (Clontech). Resin was washed three times with
20 mM Tris, pH 8, 250 mM NaCl, 5 mM DTT and the GST
Materials and Methods
Expression of GST-CKIe, GST-CKIa and CKI mutant
proteins
GST-CKIe was expressed in E. coli in Terrific Broth for
18 hours at 16uC. Cells were grown to OD600 = 2 in baffled flasks
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CKIe Interactions
fusion protein was eluted with 15 mM glutathione in the same
buffer. Protein was dialyzed overnight against 20 mM Tris, pH 8,
50 mM NaCl, 5 mM DTT at 4uC and was applied to 1 or 5 mL
HiTrap Q sepharose column (Amersham, GE Healthcare).
Protein was eluted by ion exchange using 20 mM Tris, pH 8,
800 mM NaCl, 1 mM DTT. Fractions containing the cleanest
fusion protein were frozen in liquid nitrogen and stored at 280uC.
For generation of CKIe without the GST-tag, ion-exchange
fractions were pooled and re-bound to glutathione resin. After
being washed with 20 mM Tris, pH 8, 250 mM NaCl, 5 mM
DTT, the resin was incubated over night with TEV protease at
room temperature, in the same buffer, with 1 mM EDTA and
1 mM DTT added. The supernatant from the overnight cleavage
contained CKIe, which was either frozen in liquid nitrogen and
stored at 280u C, or was further purified by gel filtration on a
Sephadex 200SE column. Gel filtration fractions were pooled and
concentrated on VivaSpin MWCO 10,000 filtration devices (GE
Healthcare) and were frozen in liquid nitrogen and stored at
280uC.
GST-CKIa was expressed in E. coli, in Luria Broth for three
hours at 18uC. Cells were grown to OD600 = 1, and were induced
with 0.5 mM IPTG. Mutants of CKIa and CKIe were expressed
at 18–24uC, for 3–18 hours, depending on the construct. Cells
were grown to OD600 = 1, and were induced with 0.5 mM IPTG.
GST was expressed in E. coli from the pGex-4T1 plasmid as in
[65].
with 100 mM EDC (Pierce) for three hours at room temperature.
The crosslinked protein was dialyzed against two changes of
20 mM Tris, pH 7.5, 50 mM NaCl for 5 hours at 4uC. Protein
was concentrated by evaporation to 10 mL and resuspended in
25 mM Ammonium bicarbonate (pH 7.8) in 6 M Urea. CKIe
that was dephosphorylated prior to trypsinization was incubated
with SAP for 1.5 hours at 30uC after dialysis; tryptic peptides that
were dephosphorylated were also incubated with SAP for
1.5 hours at 30uC, immediately after tryptic digestion. Peptides
were desalted on C-18 spin columns (The Net Group, Inc) and
infused into a hybrid-LTQ-Orbitrap mass spectrometer via
electrospray ionization (ESI). Crosslinked peptides were identified
by using the open modification search tool POPITAM [67]. Posttranslational modifications that corresponded to peptides were
searched to identify intramolecular crosslinks.
Supporting Information
Figure S1 Tandem mass spectrum of cross-linked peptide from
dephosphorylated CKIe with a precursor mass of 2686.3876
daltons. All labeled fragment ions were identified with more than
10 ppm mass accuracy, written in red above the ion peak. Ions
that
are
labeled
in
green
are
from
peptide
DVKPDNILMGLGKK and ions labeled in blue are from peptide
NPEDLDRER. Solid horizontal bars mark ions whose relative
abundance was greater than the scale at left.
Found at: doi:10.1371/journal.pone.0004766.s001 (1.02 MB EPS)
Plasmids and GST-CKIe mutants
Figure S2 Tandem mass spectrum of cross-linked peptide from
phosphorylated CKIe with a precursor mass of 3982.0332 daltons.
All labeled fragment ions were identified with more than 10 ppm
mass accuracy, written in red above the ion peak. Ions labeled in
green are from peptide KMSTPIEVLCK and ions labeled in blue
are from peptide FGAARNPEDLDRERREHDREER. Solid
horizontal bars mark ions whose relative abundance was greater
than the scale at left.
Found at: doi:10.1371/journal.pone.0004766.s002 (0.73 MB EPS)
mPer1-myc was a generous gift from D. Virshup. xDsh-HA is as
in [22]. mAxin-myc is from [66]. XCKIe -GST was created by
inserting CKIe (EST) from Xenopus laevis into pGEX-4T1. XCKIaGST was made by inserting CKIa (EST) from Xenopus laevis into
pGEX-4T1. Mutagenesis of CKIa and CKIe was performed
according the Stratagene QuikChange protocol. GST-CKIa2.e
-tail was constructed such that the CKIa sequence ends at residue
296, and the CKIe sequence begins with CKIe residue 288.
Figure S3 Tandem mass spectrum of phosphorylated peptide
MGQLRGSATRALPPGPPAGAAPNR with a precursor mass of
2502.7794 daltons. All labeled fragments were identified with
SEQUEST, and the spectrum shown was taken directly from the
SEQUEST results. Charge states are indicated by +, and
phosphorylated residues are represented by lower case letters in
the peptide sequence.
Found at: doi:10.1371/journal.pone.0004766.s003 (0.62 MB EPS)
GST pull-down experiments
Pull-down experiments were as performed in [65] with the
following differences. For most experiments, cell pellets were
thawed, sonicated and the lysate was applied to glutathione resin
for 10 minutes at room temperature. Beads were washed three
times with wash buffer (20 mM Tris, pH 7.5, 150 mM NaCl,
0.5% Nonidet P-40, 1 mM DTT). Binding reactions were done at
4u C for 40 minutes in wash buffer. After binding, beads were
washed three times for 5–10 minutes with wash buffer prior to
addition of sample buffer. Except where noted, all full-length
CKIe and CKIaRe constructs were dephosphorylated using
Shrimp Alkaline Phosphatase (Fermentas) for 1.5 hours at 30uC
after binding to glutathione sepharose. The bound protein was
then washed three times to remove traces of phosphatase. For
experiments requiring hyperphosphorylated CKIe, GST-CKIe
was bound to glutathione and allowed to autophosphorylate in the
presence of 10 mM MgCl2 and 2 mM ATP for 1.5 hours. Except
where noted, all experiments that required full-length CKIe were
run with chromatographically purified protein (including crosslinking and mass spectrometry experiments) and all other
experiments were run with partially purified protein from crude
E. coli lysate.
Figure S4 Tandem mass spectrum of phosphorylated peptide
ISASQASVPFDHLGK with precursor mass of 1636.5736
daltons. All labeled fragments identified with SEQUEST, and
the spectrum shown was taken directly from the SEQUEST
results. Charge states are indicated by +, and phosphorylated
residues are represented by lower case letters in the peptide
sequence.
Found at: doi:10.1371/journal.pone.0004766.s004 (0.60 MB EPS)
Acknowledgments
We thank David Virshup for suggesting the investigation of Period, and
Benjamin Martin for reading this manuscript.
Author Contributions
Crosslinking and mass spectrometry
Conceived and designed the experiments: CLD EZN DRG DK.
Performed the experiments: CLD EZN. Analyzed the data: CLD EZN
DK. Contributed reagents/materials/analysis tools: EZN DRG DK.
Wrote the paper: CLD DK.
Purified CKIe (2 mg/mL) was incubated with 10 mM MgCl2
and 10 mM ATP for 1.5 hours, at 30uC. CKIe was diluted 4-fold
in 100 mM potassium phosphate buffer (pH 7.5) and incubated
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CKIe Interactions
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